Chronic obstructive pulmonary disease (COPD), the third leading cause of death in the U.S, is strongly influenced by cigarette smoking and genetic predisposition. The primary objectives of this proposal are to identify functional genetic variants in two COPD genome-wide association (GWAS) loci, HHIP and FAM13A regions, and to define the molecular mechanisms by which these functional variants alter gene expression and cellular phenotypes relevant to COPD. We hypothesize that functional variants in these two loci are regulatory variants that can be measured by quantifying enhancer activity in human bronchial epithelial cells. Furthermore, we hypothesize that functional variants, through differential bindin to specific transcription factors, regulate HHIP or FAM13A gene expression, thus affecting cellular sensitivity to smoke-induced cell injury. To test these hypotheses, in the R21 phase of the project, we will apply recently developed massively parallel reporter assays (MPRA) to identify functional variants that show allele-specific enhancer activity in Beas-2B cells exposed to either room air or cigarette smoke (CS), followed by validation in reporter assays. We will also confirm allele- specific open chromatin in primary human bronchial epithelial cells (HBE) using FAIRE, and we will assess long-range interactions of identified regulatory elements with the relevant promoters using chromosome conformation capture assays. In the R33 phase of the project, we will determine the role of the functional variants in smoke-induced cell death in both Beas-2B cells and HBE. Subsequently, we will apply an siRNAs library targeting 1529 known human transcription factors (TFs) to identify TFs that bind to the identified enhancer elements with differential affinity to genetic variants (by EMSA and ChIP), alter enhancer activity (using co-transfection and reporter assays), and regulate HHIP or FAM13A expression (by RT-PCR following gene silencing) in Beas-2B cells. To demonstrate the clinical relevance of these mechanistic studies, we will perform in vivo validation in HBE cells from smokers with established COPD vs. smoking controls. Ultimately, these functional characterizations will identify biological pathways with potential therapeutic implications.